Current protocols for the isolation of microvesicles (MVs) and exosomes, which in the main focus on differential centrifugation, vary considerably (1). In an attempt to set a new standard, we describe a filtration protocol for isolating phosphatidylserine-positive MVs (larger than 100 nm in diameter) and exosomes. The key preparative step to successfully isolate both MVs and exosomes to a high degree of purity was a gentle sonication to break up exosome clumps. Filtration through a 100 nm pore size Millipore filter allowed for collection of exosomes in the filtrate. The larger MVs could then be recovered from the filter. Annexin V-PE MVs were sized and quantified using Polysciences Polybead Microspheres (200 nm) and BDTrucount tubes, respectively on a FACS CaliburTM flow cytometer. The normal reference range from normal human donors was found to be 0.51-2.82 x105 MVs/ml. Freeze/thawing of samples had little effect on MV counts and with age MV levels seemed only marginally reduced. Fasting status also affected MV levels, appearing up to 3-fold higher in fasting individuals. Smokers had lower MV counts and nicotine reduced MV release from THP-1 cells.
|Publication status||Published - 13 Sept 2012|
|Event||Microvesiculation and Disease, a Biochemical Society Focused Meeting: Microvesiculation and Disease - London Metropolitan University, London, United Kingdom|
Duration: 13 Sept 2012 → 14 Sept 2012
|Conference||Microvesiculation and Disease, a Biochemical Society Focused Meeting|
|Period||13/09/12 → 14/09/12|